Currently individuals with antibodies reactive with human immunodeficiency virus (HIV) are determined by immunoassays of the conventional sandwich ELISA format. These assays are comprised of an immobilized viral lysate that is contacted with blood or serum suspected of containing HIV antibodies. While the existing commercial tests appear to have significantly diminished the transmission of HIV via blood products, viral lysate-based tests have several significant disadvantages. These disadvantages include, but are not limited to, the need to grow and handle large quantities of live infectious virus; the possibility that live virus might be incorporated into test materials; the variable nature of the resulting lysate; and the substantial number of false positive and false negative results which necessitate additional combinatory testing, such as by Western blot.
In commonly owned U.S. Pat. No. 4,629,783 to Cosand, incorporated herein by reference, several synthetic peptides are disclosed which mimic immunologically reactive viral proteins epitopes. Among them, a peptide comprising a sequence of 26 amino acids of gp41, designated peptide 39, was found to be reactive with all known HIV-1 positive sera tested and was not reactive with any negative sample tested. Subsequently, several groups have recognized the importance of the peptide 39 region defined by Cosand as containing a dominant antigenic determinant. See Wang, J. J. G. et al. Proc. Natl. Acad. Sci. 83:6159 (1986), Shoeman, R. L. et al. Analyt. Biochem. 161:370 (1987), Gnann, J. W. et al. J. Virol. 61:2639 (1987), and Gnann, J. W. et al. J. Infect. Dis. 156:261 (1987).
Synthetic peptides offer significant advantages over viral lysates. Advantages include, but are not limited to, specificity, purity, ease of production, etc. Potential disadvantages include the need to use more than one peptide to detect all viral strains; and peptides being inherently small in size, loss of immunological reactivity upon immobilization on a solid phase.
Small peptides used as immobilized antigens in immunological assays are generally synthesized by solid phase methods, wherein each amino acid which comprises the peptide is added sequentially in a protected form while the growing peptide chain is immobilized on an insoluble resin support. During the synthetic process side chains of certain amino acids comprising the growing chain are protected, blocked by a variety of chemical groups which remain until the completed peptide is removed from the insoluble resin support, usually by reaction with concentrated hydrofluoric or trifluroacetic acid. During cleavage the majority of the protective groups are removed and the completed peptide is purified by any of a variety of methods well known in the art.
Crude or purified peptide is then immobilized onto a solid phase by a variety of methods, including direct conjugation, conjugation to a soluble non-immunologically reactive protein or other inert molecule, or by direct adsorption onto a solid phase. U.S. Pat. No. 4,629,783 discloses examples of several of these methods.
Peptides which mimic epitopes from the gp36 protein of HIV-2, a glycoprotein believed to be comparable in retroviral function to gp41 of HIV-1, are disclosed in co-owned U.S. patent applications U.S. Ser. No. 030,403 and U.S. Ser. No. 035,408, filed Mar. 25, 1987 and Apr. 7, 1987, respectively, herein incorporated by reference. Among these, a peptide designated 41-2-1, comprised of 26 amino acids encoded within a region similar to that of peptide 39 in HIV-1, was found to be reactive with all known HIV-2 positive sera tested and was not reactive above appropriate cut-off levels with negative samples which were tested. A shortened version of peptide 41-2-1, designated 41-2-3, comprising amino acids residues from the carboxyl end of peptide 41-2-1, was also found to be reactive with the HIV-2 sera and in some cases performed better than peptide 41-2-1.
Peptides 39, 41-2-1 and 41-2-3 each contain within their amino acid sequence two cysteine residues. The presence of cysteine residues within a peptide sequence allows for the formation of inter- and intra-molecular disulfide bonds during purification, immobilization, and upon long term storage of a deprotected peptide. Therefore, such peptide compositions are usually immobilized on a solid phase as a mixture of a variety of oxidative forms, including monomer, dimer and polymers of various sizes. Precautions are generally not taken to control the oxidative form of peptides immobilized on a solid phase. Rosen, J. I. et al. (W087/06005) recognized that certain peptides derived from the highly antigenic region of HIV1, identified by Cosand, supra, which contained more than one cysteine residue were immobilized as a mixture of various oxidative forms and suggested that the oxidative form of a peptide, particularly polymers, may be of importance to the reactivity of certain peptides. Rosen et al. disclose that in some cases the cyclic form of certain peptides is less efficient at binding to solid surfaces than polymeric forms. The variety of oxidative forms of the peptides may be a source of variability in sensitive immunoassay and this may also influence the results based on those assays.
Accordingly, there exists a significant need in the art for peptide compositions with improved reactivity, and which provide as few false-negative and false-positive results as possible. The present invention fulfills these and other needs.